The present invention relates to a breath test for the diagnosis of bacterial infection and, more particularly, to a breath test for the diagnosis of infection by Chlamydia species.
Chlamydia species are bacteria which are responsible for a number of different infections in humans. Chlamydia trachomatis is a sexually transmitted disease. Recently, Chlamydia pneumoniae has been implicated in coronary heart disease, specifically in the development of atheroma, which is diseased tissue that can block the arteries, potentially leading to a myocardial infarction. Evidence of such an involvement has been growing and includes the detection of DNA from Chlamydia pneumoniae in samples of coronary tissue taken from patients with coronary heart disease (described in "Can You Catch a Heart Attack?" by P. Brown, New Scientist, June 1996, p. 38-42). Furthermore, a high proportion of patients with acute myocardial infarction also had an acute C. pneumoniae infection (F. Blasi et al., "A Possible Role for Chlamydia pneumoniae Infection in Acute Myocardial Infarction Onset", Abstracts from the Third European Chlamydia Meeting, Sep. 11-14, 1996, Vienna, Austria, p. 220). These and other results have generated interest in the diagnosis of infection by Chlamydia species.
The life cycle of Chlamydia species, particularly of Chlamydia pneumoniae, increases the difficulty of detecting these bacteria. Essentially, these bacteria have two parts in their life cycle. In one part, these bacteria enter a host cell and live as an intracellular reticular body. In the second part, the bacteria leave the host cell and live as an extracellular elementary body. Thus, the bacteria can "hide" within the host cell, inaccessible to the immune system of the host, and then leave the host cell in order to infect other tissues of the host. Since the entrance of the bacteria into the host cell requires an alteration in the integrity of the latter cell membrane, such an infection of individual host cells could cause a lesion in the host tissue. Thus, not only does such an infection make Chlamydia species harder to detect, it also could lead directly to damage of the host tissue, such as a coronary artery. Clearly, rapid, accurate and non-invasive methods of detection of Chlamydia species are necessary.
Unfortunately, currently available methods to diagnose an infection by a Chlamydia species, particularly by Chlamydia pneumoniae, are invasive and difficult to perform, and cannot measure Chlamydia activity in "real time". That is, there is a significant delay between the time the Chlamydia activity takes place, and the time such activity is measured by the test.
For example, PCT Patent No. 9222819 to Kuo (hereinafter referred to as "Kuo") discloses a method of detecting a first marker associated with Chlamydia pneumoniae in a biological sample, and of detecting a second marker associated with arterial granuloma in the same sample. The Chlamydia marker includes the concentration of lipids from Chlamydia pneumoniae in a serum or tissue sample. Such a method has the disadvantage of requiring a serum or tissue sample, both of which are biohazards. Furthermore, this test examines two markers, which requires two separate measurements. Certain of these measurements are made in a static sample in vitro, rather than being made in real time in vivo. Thus, the concentration of such a marker is not necessarily proportional to the actual activity of Chlamydia bacteria in a subject, and more importantly, a significant delay must take place between the time the activity takes place, and the time the measurement is made.
Examples of such markers include antibodies raised against, and lipids released from, Chlamydia pneumoniae. The disadvantage of these types of markers is that they do not reflect current activity of the bacteria. The presence of antibodies, for example, can indicate a current infection, or an infection which occurred and was resolved previously. Thus, it is difficult to determine the level of the present activity of the bacteria, if any.
Those markers which are described as being measured in vivo have other disadvantages. For example, the disclosure mentions a radiolabelled antibody against Chlamydia pneumoniae which is delivered through a catheter to the heart. This procedure is very invasive and does not allow the level of bacteria to be easily measured, since the antibody would not necessarily be able to bind to bacteria in the intracellular portion of their life cycle. Furthermore, such a procedure also cannot directly measure the level of activity of the bacteria themselves in real time.
PCT Patent No. 9000061 to Saikku discloses a method of detecting the presence of Chlamydia in vitro by using a sample of blood, urine or tissue biopsy from the heart. Obtaining a tissue biopsy is clearly invasive, and thus disadvantageous. Even using a urine sample is somewhat disadvantageous, since it requires handling of a bodily fluid which is potentially biohazardous. Furthermore, this method uses antibodies to detect Chlamydial antigens, or alternatively measures the level of antibodies against Chlamydia, rather than measuring the activity of the bacteria themselves. Not only is such a method indirect, but it also creates a delay between the time the Chlamydia activity occurs in the subject, and the time the measurement is made. Also, the presence of antibodies indicates only that an infection by Chlamydia species occurred at some point in the past, not that such an infection is current. Furthermore, no mention is made of an in vivo method of measurement. Thus, all of the currently available methods of diagnosing Chlamydia species are disadvantageous.
However, rapid and non-invasive methods of detecting infection by Helicobacter pylori in the gastrointestinal tract have been described. These methods involve administering a substrate to a subject and then analyzing the exhaled breath of the subject for the presence of a hydrolysis product or products, which indicate the presence of Helicobacter pylori in the gastrointestinal tract. For example, U.S. Pat. No. 4,830,010 to Marshall (hereinafter referred to as "Marshall") describes a method of detecting Helicobacter pylori by orally administering isotopically-labelled urea to a subject. Helicobacter pylori produces a large quantity of the enzyme urease, which hydrolyzes urea to form carbon dioxide and ammonia. Either one or both of these hydrolysis products can have the isotopic label. At least one isotopically-labelled product is then exhaled by the subject and can be detected in the exhaled breath of the subject by an appropriate measuring instrument. Thus, the breath test for diagnosing Helicobacter pylori is rapid, easy to perform and relatively non-invasive. Unfortunately, no such breath test is currently available for the diagnosis of Chlamydia species.
There is thus a widely recognized need for, and it would be highly advantageous to have, a breath test for the detection of Chlamydia species in a subject, which is relatively non-invasive, and which can measure the activity of Chlamydia bacteria with relatively little delay.